Conventional vaccine adjuvants with nanoparticle components such as oil-in-water emulsions or aluminum salts do not promote effective Th1-type cellular immune responses, which are critical for complex diseases lacking effective vaccines such as malaria, tuberculosis, and amebiasis. The latter is a neglected enteric disease caused by the protozoan parasite Entamoeba histolytica (a biodefense category B pathogen) and accounts for significant disease burden with some 50 million annual infections worldwide, with the greatest burden in the developing world. This proposal describes the development and physicochemical characterization of nanoliposomes containing a combination of synthetic Toll-like receptor (TLR) ligands, and the evaluation of their ability to enhance amebiasis vaccine immunogenicity and protective efficacy. The efficacy of live attenuated vaccines may in part be attributed to their capacity to trigger multiple pathogen recognition receptors such as TLRs. Nevertheless, for modern recombinant or inactivated vaccines, the only TLR ligand-based adjuvant used in an FDA-approved vaccine (i.e. Cervarix(R)) consists of a single TLR ligand. However, appropriate nanoformulation of TLR ligand combinations may promote synergistic activation of immune responses, including Th1-type cellular immunity. In the proposed work, we will employ high pressure homogenization to manufacture stable nanoliposomes containing a synthetic TLR4 ligand (GLA) and a synthetic TLR7/8 ligand (3M052). The nanoliposomes will be manufactured at ~65 and 100 nm diameters as well as with different surface characteristics (PEGylation length and concentration). Nanoliposomes demonstrating acceptable in vitro physicochemical stability as well as compatibility with a recombinant amebiasis antigen (LecA) will next be evaluated for their ability to enhance Th1-type immune responses to LecA in a mouse model after subcutaneous administration. Furthermore, the immunization regimen will be adapted to include complementary intranasal administration in order to assess the ability to elicit mucosal IgA responses (which are also correlated with protection against amebiasis) while maintaining strong Th1-type cellular responses. Finally, the nanoliposome adjuvants and immunization regimens demonstrating optimal immunogenicity profiles will be evaluated in an amebiasis mouse challenge model for their protective efficacy. The broad implications of the project include demonstrating that protective Th1-type immunity can be achieved through appropriate nanoformulation of a combination of TLR ligands in a biocompatible and stable liposomal vehicle. This approach could be applied to accelerate vaccine development for many other biodefense pathogens. Successful vaccines developed for amebiasis or other indications requiring Th1-type immune responses such as malaria or tuberculosis would result in significant public health benefits.